Bone elongation in children occurs by endochondral ossification in cartilaginous growth plates at the ends of long bones. The parent R01 entitled In vivo Imaging of Growth Plate Dynamics funds a project using multiphoton microscopy to visualize molecular transport and cellular dynamics in growth plate cartilage of living mice. These studies have led to specific questions concerning oxygen levels within growth cartilage, and their relationship both to cellular metabolism during chondrogenic differentiation and to signaling cascades leading to vascular invasion of cartilage from metaphyseal endothelial cells. Although for more than fifty years experimental data have suggested that there is an O2 gradient within the growth plate extracellular matrix (ECM) that has an essential role in chondrocytic differentiation, definitive measurements still are inconclusive with conflicting results depending upon the methodology and the experimental system. What contributes to the ambiguity concerning O2 delivery to, and movement within, the growth plate cartilage ECM is that researchers are unable to directly map O2 concentrations within and around the region in vivo. Our BIRT proposal describes a highly interdisciplinary effort to produce oxygen-sensitive phosphors based on ruthenium complexes that are bright, sensitive and stable. Tailored for multiphoton microscopy, they will yield rapid micron-resolved images of O2 tension relatively deep into and around the growth cartilage of mice in vivo. Candidate sensors will be tested not only for their photophysical properties, but also for how they interact and function within tissue. Probe developments will be applicable both to confocal and multiphoton microscopy, and potentially whole animal "molecular imaging" systems. Our goal is to relate microenvironmental O2 levels to specific stages of chondrocytic differentiation, and to correlate O2 gradients within the ECM with O2 availability levels in three different surrounding vascular routes. The multidisciplinary team assembled for this BIRT award combines the expertise of a chemist with experience in designing oxygen-sensing probes, a nanotechnologist with skills in designing non-toxic delivery systems, a physicist with expertise in using multiphoton microscopy for radiometric measurements and in vivo imaging systems, and a biologist whose research has focused on cellular dynamics of the chondrocytic differentiation cascade in the postnatal animal. [unreadable] [unreadable] [unreadable]